This invention relates to a modified bioluminescent system comprising a flourescent molecule covalently linked with a photoprotein allowing the transfer of energy by Chemiluminescence Resonance Energy Transfer (CRET). This invention also relates to the use of the modified bioluminescent system in in vivo and in vitro assays.
Calcium is implicated in the regulation of a great variety of intracellular processes (1). Several techniques are most commonly used for intracellular Ca++ monitoring. Patch-clamp and Ca++ selective microelectrodes give cumulative measurements of Ca++ fluxes in a restricted number of cells. On the other hand, intracellular [Ca++] dynamics in large populations of cells can be visualized with fluorescent probes (2). Genetic tools could provide new methods for Ca++ monitoring.
Two groups of genetic Ca++ probes are at present available. The first category uses the principle of Fluorescence Resonance Energy Transfer (FRET) between two variants of the green fluorescent protein (GFP). The two GFP are covalently linked by a calmodulin binding sequence alone or in combination with calmodulin so that intramolecular FRET does (3) or does not (4) occur in response to Ca++ influx. The second category is composed by bioluminescent proteins, such as aequorin (5, 6). The active protein is formed in the presence of molecular oxygen from apoaequorin (189 amino acids) and its luciferin, coelenterazine (Mr 423) (7).
The binding of Ca++ to aequorin, which has three EF-hand structures characteristic of Ca++ binding sites, induces a conformational change resulting in the oxidation of coelenterazine via an intramolecular reaction. Moreover, the coelenteramide so produced is in an excited state, and blue light (max: 470 nm) is emitted when it returns to its ground state (8). Such a bioluminescent genetic marker presents the advantage over Ca++-sensitive fluorescent dyes of being easily targeted to specific cells and in subcellular compartments with appropriate regulatory elements and peptide signals (9). The bioluminescent process does not require light excitation like fluorescent probes or proteins, and thus does not induce autofluorescence, photobleaching and biological degradation problems. Furthermore, aequorin is not toxic, does not bind other divalent cations and does not interfere with the [Ca++], buffer system even when microinjected at high concentrations. Its low affinity for Ca++ (Kd=10 (xcexcM) is probably responsible for this and makes aequorin a good sensor in the range of biological [Ca++] variations.
Although providing a good ratio of signal over background, aequorin signals are very difficult to detect because of aequorin""s low light quantum yield, that is, the number of emitted photons per protein that bind Ca++. In the jellyfish, Aequorea victoria, from which aequorin has been isolated (10), the protein is associated with the GFP (11). After Ca++ binding, the energy acquired by aequorin is transferred from the activated oxyluciferin to GFP without emission of blue light. The GFP acceptor fluorophore is excited by the oxycoelenterazine through a radiationless energy transfer. Then, a green light (max, 509 nm) is emitted when the excited GFP returns to its ground state (12).
Such intermolecular radiationless energy transfer is not unusual in bioluminescence and has already been shown to increase the quantum yield of the bioluminescent process in Renilla, another coelenterate (13). The gain measured in vitro ranges from 3 to 5 fold (14). It is possible to reconstitute in vitro the Renilla system and obtain the spectral shift with low equimolar concentrations of its components because the luciferase and the green fluorescent protein bind together (14).
In the Aequorea system, binding between purified photoprotein and GFP does not occur in solution, even when present at high concentrations (15). In vivo, energy transfer occurs because of the high concentration of GFP. It can be obtained in vitro through the co-adsorption of aequorin and GFP on DEAE cellulose membranes (15). The Fxc3x6rster equation shows that the efficiency of this process depends on several conditions described in the case of FRET. The emission spectrum of the donor must have the greatest overlap with the excitation spectrum of the acceptor. The energy transferred is also strongly dependent on the geometry, in particular, the relative orientation and distance of the two dipoles and modulated by their respective motion (16).
An aim of this invention is to develop a dual reporter gene combining properties of Ca++-sensitivity and fluorescence of aequorin and GFP, respectively. The fusion protein, which can be detected with classical epifluorescence, can be used to monitor calcium activities. The configuration of the molecules of the invention increases their overall turnover and allows an efficient intramolecular Chemiluminescence Resonance Energy Transfer (CRET). As a result, the quantum yield of aequorin appears to be higher. This invention shows that physiological calcium signals can be visualized in single eukaryotic cells with an intensified CCD camera. Other constructs described here target the fusion protein to the neurite membrane.
This invention thus provides a modified bioluminescent system comprising a fluorescent molecule covalently linked with a photoprotein, wherein the link between the two proteins has the function to stabilize the modified bioluminescent system and allow the transfer of the energy by Chemiluminescence Resonance Energy Transfer (CRET). In a preferred embodiment, the bioluminescent system comprises an GFP protein covalently linked to a aequorin protein, wherein the link between the two proteins has the function to stabilize the modified bioluminescent system and to allow the transfer of the energy by Chemiluminescence Resonance Energy Transfer (CRET).
In one embodiment of a modified bioluminescent system according to the invention, the bioluminescent system comprises an GFP protein covalently linked to an aequorin protein, wherein the link between the two proteins is constituted by at least 5 amino acids and optionally at least 5 amino acids and at least one copy of 9 amino acids. The link has the function of stabilizing the system and allowing the transfer of energy by Chemiluminescence Resonance Energy Transfer (CRET).
In a preferred embodiment, the bioluminescent system comprises a GFP protein covalently linked to an aequorin protein, wherein the link between the two proteins is preferably constituted by at least 5 amino acids and five copies of 9 amino acids and has the function of stabilizing the system and allowing the transfer of energy by Chemiluminescence Resonance Energy Transfer (CRET).
The two proteins can be separate or together functional. In addition, the modified bioluminescent system can be calcium sensitive and/or light sensitive.
This invention also provides a method of screening in vitro a change in a physical, chemical, biochemical, or biological condition. The method comprises:
a) providing in different samples a bioluminescent system according to the invention in a reaction system containing an analyte of interest;
b) measuring whether light is produced; and
c) detecting a change based on the production of light.
Further, this invention provides a method of screening in vivo a change in a physical, chemical, biochemical, or biological condition. The method comprises the steps of:
a) administering to a mammal an acceptable composition comprising a bioluminescent system according to the invention;
b) detecting whether light is produced; and
c) optionally measuring ionic concentration of calcium flux.
In addition, this invention provides a composition comprising a purified polypeptide, wherein the composition has the functional characteristics of binding calcium ions and transmitting measurable energy, said energy depending on the quantity of calcium bound and on the quantity of polypeptides in said composition in absence of any light excitation.
In addition, this invention provides a purified polypeptide having the amino acid sequence of SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; and SEQ ID NO: 6.
In other embodiments, this invention provides a polynucleotide having the sequence of SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11; and SEQ ID NO: 12.
This invention also provides a culture as deposited at the C.N.C.M. and containing the plasmid No. I-2507; the plasmid No. I-2508; the plasmid No. I-2509; the plasmid No. I-2510; the plasmid No. I-2511; the plasmid No. I-2512; or the plasmid No. I-2513.
Further, this invention provides a peptide linker having the function after translation to approach a donor site to an acceptor site in optimal conditions to permit a direct transfer of energy by chemiluminescence in a purified polypeptide according to the invention. The nucleotide linker can have, for example, the nucleotide sequence of SEQ ID No: 13; SEQ ID No: 14; SEQ ID No: 15; SEQ ID No: 16, or SEQ ID No: 17. The peptide linker can comprise at least 5 amino acids and comprising the amino acid sequence of SEQ ID No: 18; SEQ ID No: 19; SEQ ID No: 20; SEQ ID No: 21, or SEQ ID No: 22.
A kit for measuring the transfer of energy in vivo or in vitro contains at least one of the polypeptides according to the invention or the polynucleotide according to the invention and the reagents necessary for visualizing or detecting the said transfer in presence or in absence of a molecule of interest.
In another embodiment, the invention provides a fusion protein of the formula:
GFP-LINKER-AEQ; 
wherein GFP is green fluorescent protein; AEQ is aequorin; and LINKER is a polypeptide of 4-63 amino acids, preferably 14-50 amino acids.
The LINKER can comprise the following amino acids:
(Gly Gly Ser Gly Ser Gly Gly Gln Ser [SEQ ID NO: 25])n, wherein n is 1-5. Preferably n is 1 or n is 5. LINKER can also include the amino acid sequence Ser Gly Leu Arg Ser [SEQ ID NO: 26].
Another fusion protein for energy transfer from aequorin to green fluorescent protein by Chemiluminescence Resonance Energy Transfer (CRET) following activation of the aequorin in the presence of Ca++ has the formula:
GFP-LINKER-AEQ; 
wherein GFP is green fluorescent protein; AEQ is aequorin; and LINKER comprises the following amino acids:
(Gly Gly Ser Gly Ser Gly Gly Gln Ser [SEQ ID NO: 25])n, wherein n is 1-5; and wherein the fusion protein has an affinity for Ca++ ions and a half-life of at least 24 hours. The LINKER can include the amino acid sequence Ser Gly Leu Arg Ser [SEQ ID NO: 26]. In addition, the fusion protein can further comprise a peptide signal sequence for targeting the fusion protein to a cell or to a subcellular compartment.
This invention also provides polynucleotides encoding fusion proteins as described above.